This Application is a 371 of PCT/FR98/01074 filed May 28, 1998, which claims priority from France 97/06667 filed May 30, 1997.
The present invention relates to a novel approach for treating cystic fibrosis which involves chemotherapy, in particular anticancer chemotherapy.
Cystic fibrosis is a genetic disease which is expressed in particular in the lungs and which is due to a defect in the gene encoding the CFTR (standing for xe2x80x9cCystic Fibrosis Transmembrane Conductance Regulatorxe2x80x9d) protein, which is a protein which is able to participate directly or indirectly in the transport of chloride ions across the cell membranes.
In a general way, the CFTR protein belongs to the ABC (standing for xe2x80x9cATP Binding Cassettexe2x80x9d) transporter family, which is a very extensive family of proteins whose members are found both in eukaryotes and in prokaryotes. In general, these proteins are active transporters which hydrolyze ATP in order to supply the chemical potential which is required for their transport function. Thus, in eukaryotes, they transport various types of molecule across the cell membranes. Molecules which are capable of being transported and which may be mentioned include ions, vitamins, peptides, sugars and medicinal substances or other drugs. Their overall organization has common features: they generally comprise a transmembrane (TM) region, which is involved in selecting the chemical entity to be transported, and a nucleotide-binding domain which hydrolyzes the ATP in order to supply the chemical potential which is required for the transport (NBF standing for xe2x80x9cNucleotide Binding Foldxe2x80x9d).
The genes which encode these proteins are often derived from the fusion of two genes of analogous structure.
The structure of the corresponding proteins is then generally as follows:
(TM1)-(NBF1)-(TM2)-(NBF2)
The CFTR protein constitutes a 1480-amino-acid-containing member of a subfamily of the ABC transporter family termed the MRP/CFTR subfamily. In addition to the CFTR protein, this subfamily contains the human MRP protein. Specifically, the two proteins exhibit a sequence similarity of approximately 50%. The MRP (standing for xe2x80x9cMulti-drug Resistance associated Proteinxe2x80x9d) is known to be involved in phenomena of multiresistance to the medicaments which are used in cancer chemotherapy (M. Dean, R. Allikmets, Current Opinion in Genetics and Development, 5, 779-785, 1995). The CFTR protein is also structurally very close to another member of the MRP/CFTR subfamily, i.e. the protein YCF1 (standing for xe2x80x9cYeast Cadmium Resistance Factor 1xe2x80x9d), which confers on the yeast Saccharomyces cerevisiae the phenotype of resistance to cadmium ions (Tommasini et al., PNAS, 93, 6743-6748, 1996). As well as their similarity in structure, the MRP and YCF1 proteins function in a similar way: they export molecules and ions in the form of their adducts with glutathione (G. J. Zaman et al., PNAS, 92, 7690-7694, 1995).
The CFTR protein also displays not insignificant structural similarities with the yeast STE6 protein, which transports the pheromone xe2x80x9cfactor axe2x80x9d in the yeast Saccharomyces cerevisiae (J. L. Teem et al., Cell, 73, 335-346, 1993) and with the human MDR (standing for xe2x80x9cMulti-drug Resistance Proteinxe2x80x9d) protein, which is known, like MRP, to be involved in the phenomena of multiresistance to anticancer medicaments. The human MDR protein and the STE6 protein belong to another subfamily of ABC transporters, termed the MDR/TAP subfamily.
Thus, the general adherence of the CFTR protein to the ABC transporter family, and its function of transporting chloride ions, are now well known.
In a patient suffering from cystic fibrosis, the CFTR protein is mutated. While more than 600 mutations have been recorded, the mutation involved is, in approximately 70% of cases, the deletion of a phenylalanine in position 508 in the NBF1 part (xcex94F508) of the overall structure of the protein. It appears that this mutation results in a defect in the folding of the protein which is then destroyed within the cell without completing its post-translational maturation (Riordan J. R., Rommens J. M., Kerem B. S., Alon N., Rozmahel R., Grzelczack Z., Zielenski J., Lok S., Plavic N., Chou J. L., Drumm M. L., Iannuzzi M. C., Collins F. S., Tsui L. C. (1989) Science. 245, 1066-1072). The absence of a mature or functional CFTR protein leads to a defect in the secretion of chloride ions. The so-called xe2x80x9csweatxe2x80x9d test, which measures the secretion of chloride ions, was, moreover, developed in 1953 and remains indispensable for diagnosing cystic fibrosis.
Up to now, attempts have been made to treat cystic fibrosis by gene therapy, by means of developing systems for administering to patients a nucleic acid which encodes the wild-type CFTR protein and which is transported either by viruses or by cationic lipids. Attempts have been made to administer such a DNA/cationic lipid complex to lungs of mice by the intratracheal route (Yoshimura et al., Nucleic Acid Research, 20 :3233-3240, 1992) or by means of an aerosol (Stribling et al., Proc. Natl. Acad. Sci. 89:11,277-11,281, 1992). It has also been observed that administering the CFTR-encoding gene, in a complex together with cationic lipids, to a mouse model suffering from cystic fibrosis had the effect of correcting the defect in the function of the chloride ion channel (Hyde et al., Nature 362:250-255, 1993).
Thus, the therapeutic approaches which are currently envisaged in the case of cystic fibrosis are solely aimed at the genetic defect (mutation in the gene encoding the CFTR protein), which they are endeavoring to correct. They link the efficacy of the treatment to the re-establishment of the only chloride-ion channel function which is exerted by a functional CFTR protein.
However, even though the defect in the transport of chloride ions is indeed a clinical manifestation of cystic fibrosis, other manifestations have still not been fully explained.
Thus, as an example, the organs which are chiefly affected in cystic fibrosis are those in which glutathione is secreted, in particular the liver, or those in which detoxification mechanisms are likely to involve glutathione (lungs, intestine, colon).
Furthermore, it has been noted that the inflammatory reaction is excessive in patients suffering from cystic fibrosis. A chronic inflammation of the airways is often observed in these patients. When this occurs, a very high number of neutrophilic granulocytes is present in the airways of the patients, even when there is no detectable infection. It is possible that this inflammation precedes the appearance of the chronic infection. This influx of neutrophilic granulocytes results in a massive release of free radicals and hyperoxides in the cells of the airways of the patients. Now, it is known that exocellular and intracellular glutathione plays a central role in the control of the inflammatory reaction by protecting the cells from attack by these oxidizing agents. However, this protection appears to be impaired in patients suffering from cystic fibrosis. It would therefore appear that cystic fibrosis prevents glutathione from playing its customary role.
Studies performed on the family of ABC transporter proteins have demonstrated their relative versatility. Even though each of the proteins possesses its own function, they appear to display a shared mode of operation which enables them to undertake shared functions. For example, it has been demonstrated in vitro that the human MRP protein is able to act as a substitute for YCF1 in yeast and to undertake the function of detoxifying cadmium in this organism (Tomassini et al., PNAS, 93, 6743-6748, 1996). It can also act as a substitute for the STE6 protein in yeast in order to undertake the transport of factor a in this organism (J. L. Teem et al., Cell, 73, 335-346, 1993). Similarly, an STE6 chimeric protein, in which the CFTR NBF1 domain has been substituted for the STE6 NBF1 domain, is functional and efficiently transports factor a (J. L. Teem et al., Cell, 73, 335-346, 1993). Finally, it would appear that, in vivo, the genes which encode the CFTR, MDR and MRP proteins are under coupled transcriptional controls (Trezise A. E., Romano P. R., Gill D. R., Hyde S. C., Sepulveda F. V., Buchwald M., Higgins C. F., EMBO J., 11, 4291-4303, 1992 and M. A. Izquierdo, J. J. Neezfjes, A. E. L. Mathari, M. J. Flens, G. L. Scheffer, R. J. Scheper, British Journal of Cancer, 74, 1961-1967, 1996).
The inventors have now found that it is possible, surprisingly, to use this versatility in the ABC transporter proteins to conceive of treating cystic fibrosis by chemotherapy, by administering certain types of product to the patient. Said administration would lead to the expression or overexpression of a compound which is able to play the role of a functional CFTR protein and therefore overcome the deficiencies in the mutated CFTR protein.
For this reason, the invention relates to the use, for preparing a medicament which is intended for preventing and/or treating cystic fibrosis, of at least one product whose administration to a patient suffering from cystic fibrosis leads, in said patient, to the expression or overexpression of at least one ABC transporter compound.
It is assumed that, in the case of treating cystic fibrosis, the mechanism of action of the compound which is expressed or overexpressed is such that the compound is able to act as a substitute for the CFTR protein which is defective in the patient. It is assumed that the compound then exerts a function which is normally fulfilled by the wild-type CFTR protein and which the defective CFTR protein is unable to fulfill in the patient suffering from cystic fibrosis.
Thus, products which can be used for treating cystic fibrosis are those which are able to induce, in the body of the patient, the expression or overexpression of an ABC transporter compound, which acts as a substitute for the defective CFTR protein.
Different embodiments are conceivable. According to a first embodiment, it is possible to conceive of administering products which induce the expression or overexpression of the patient""s mutated CFTR protein. This protein would then be expressed at a level which was such that it recovered its ability to function despite the presence of a mutation.
According to another embodiment, products will be selected which lead to the expression or overexpression of the MDR protein.
Furthermore, studies carried out by the inventors suggest that, in addition to its role as a chloride ion channel, the CFTR protein plays the role of a glutathione pump. This hypothesis could account for a number of clinical manifestations which are exhibited by patients who are suffering from cystic fibrosis and in whom the CFTR protein is nonfunctional.
In addition, the inventors have demonstrated the existence of a potential glutathione-binding site in the NBF1 domain of the CFTR protein, a fact which could account for the role played by glutathione in the transporter function exercised by the CFTR protein. If the CFTR protein did have a role as a glutathione pump, this could, in particular, make it possible to obtain a better understanding of the chronic inflammation which is characteristic of cystic fibrosis. This is because glutathione is a key molecule in the triggering and control of the inflammatory reaction. While it is involved in the metabolism of pro-inflammatory compounds such as the leukotrienes, it is also involved, as an agent which protects against the oxidizing agents which are released by the neutrophilic granulocytes (free radicals, hydroperoxides, etc.), in controlling these reactions.
For this reason, the invention relates, more specifically, to the use, for preparing a medicament which is intended for preventing and/or treating cystic fibrosis, of at least one product whose administration to a patient suffering from cystic fibrosis leads, in said patient, to the expression or overexpression of an ABC compound which is a glutathione transporter.
According to the invention, xe2x80x9ccompound which is a glutathione transporterxe2x80x9d is understood as being a compound which belongs to the already itemized family of ABC transporters which exercise their transport function through the agency of glutathione. It is also understood as meaning any fragment or analog of such a transporter which results from one more mutations and which retains the property of transporting through the agency of glutathione.
Glutathione is understood as being glutathione itself or its adducts with other compounds. These adducts can be natural adducts, such as the leukotrienes, or detoxification adducts with heavy metals, powerful oxidizing agents (free radicals, peroxides, etc.) or drugs.
This is because it has already been established that some proteins, in particular the human MRP protein, export drugs and medicaments, in particular anticancer products, through the agency of glutathione, either in the form of direct glutathione-drug adducts or by simultaneously or sequentially binding glutathione and the drug (G. J. Zaman et al., PNAS, 92, 7690-7694, 1995).
According to a particularly preferred embodiment, an attempt will be made to express or overexpress the MRP protein. In this case, but also in the more general case where the expressed or overexpressed compound is an ABC transporter, in particular the MDR protein, the products which can be used in accordance with the invention are preferably anticancer products of the antineoplastic type, that is to say cytotoxic anticancer agents which, for treating cancer, have to kill the target cancer cells as selectively as possible. In fact, it is known that administering these anticancer agents to patients suffering from cancer often has a tendency to give rise to phenomena of resistance to anticancer agents of the pharmacokinetic type, which phenomena are caused by the overexpression of proteins of the MRP or MDR type (M. Dean, R. Allikmets, Current Opinion in Genetics and Development, 5, 779-785, 1995; Jedlitschky et al., Cancer Research, vol. 56, Mar. 1, 1996, 988-994; Akimaru, Cytotechnology, vol. 19, No. 3, 196, 221-227; Jedlitschky, Cancer Research vol. 54 No. 18, 1994, 4833-4836; Jedlitschky et al., Anticancer drugs, vol. 5 suppl. 01, Sep. 4, 1994; Mueller et al., PNAS, vol. 91, 1994, 13033-13037; Ishikawa, J. Natl, Cancer Inst., vol 87, No. 21, 1995, 1639-1640; Leier et al. Biochemical Journal, vol. 314, No. 2, 1996, 433-437).
The invention is therefore directed towards using four large families of antineoplastic agents which are employed in the treatment of cancer, i.e. alkylating agents, intercalating agents, anti-metabolites and spindle poisons, for treating cystic fibrosis.
Alkylating agents are agents which are able to replace a proton, in a molecule, with an alkyl group. They significantly alter the structure of the DNA at the time of mitoses, whose progress they disrupt. Of the large families of alkylating agents which can be used in accordance with the invention, those which may be mentioned are nitrogen mustards, nitrosoureas, platinum derivatives, ethyleneimine derivatives, dimethane sulfonoxyalkanes, piperazine derivatives and methylhydrazine derivatives. Representative examples of nitrogen mustards are chlorambucil, cyclophosphamide, ifosfamide, estramustine, melphalan and chlormethine. Use is advantageously made of ifosfamide, in particular in combination with an intercalating agent.
The intercalating agents are agents which intercalate between the two complementary strands of the DNA, thereby blocking replication of the DNA, transcription into mRNA and protein synthesis. The large families which can be used in accordance with the invention comprise, in a nonlimiting manner, anthracyclines, anthracerediones, anthracenes, acridine derivatives, ellipticines and actinomycins. Preference is given to using anthracyclines, among which may be mentioned, in a nonlimiting manner, aclarubicin, doxorubicin, daunorubicin, epirubicin, idarubicin, zorubicin and pirabucin. Preference is given to using epirubicin, in particular in combination with ifosfamide.
A third family of compounds which can be used in accordance with the invention is represented by antimetabolites, or antagonists, or structural analogs which generally inhibit one or more steps in nucleic acid synthesis. These compounds are represented, in a nonlimiting manner, by folic acid antagonists, purine antagonists and pyrimidine antagonists. Particular mention may be made of amethopterin (methotrexate), mercaptopurine, 5-fluorouracil and cytarabine.
Another category of anticancer agents is represented by the spindle poisons, which block cell mitosis. These are anti-mitotic agents whose representative families are the epipodophyllotoxins and the vinca alkaloids. Epipodophyllotoxins which may be mentioned are teniposide and etoposide. Examples of vinca derivatives which may be mentioned are vindesine, vinorelbine, vincristine and vinblastine. Preference will be given to mentioning colchicine and its derivatives. Particularly satisfactory results have been obtained with colchicine, which is all the more advantageous since this product is known to be of low toxicity.
Finally, mention must also be made of the taxoid family (taxol, taxotere, etc.).
According to another embodiment, it is also possible to use various cytolytic agents such as, for example, bleomycin, dacarbazine, hydroxycarbamide, asparaginase, mitoguazone and plicamycin.
Mention will more particularly be made of sodium phenylbutyrate, which is a cytostatic agent which is used in diseases of the urea cycle accompanied by hepatic manifestations.
Mention will also be made, as products which can be used in accordance with the invention, of the products of the macrolide family which are known for their properties of inducing overexpression of the MDR protein, leading to the phenomenon of drug resistance (MDR). Examples of these inducers are those mentioned in the publication Seelig Eur. J. Biochem., 25, 252-261 (1998).
A number of these have already been mentioned as belonging to the anticancer agents. These inducers comprise, in particular, actinomycin D, clotrimazole, colchicine, daunorubicin, doxorubicin, epothilone A, erythromycin, eroposide, isosafrole, midazolam, nifedipine, phenobarbital, puromycin, reserpine, rifampicin, taxol, vinblastine, vincristine, cysteine methyl ester, epinephrine and farnosol.
By way of example, mention may preferably be made of azithromycin, which is administered at doses lower than those required for obtaining an antibacterial activity (Jaffe et al., Lancet 1998; 351-420).
Another example of an MDR-protein-inducing macrolide which can be used in accordance with the invention is erythromycin (Grant et al., Toxicol. Appl. Pharmacol., 1995; 133:269-76).
In a more general manner, the studies carried out by the inventors make it possible to select products which are capable of being used in the prevention and/or treatment of cystic fibrosis by properly proportioning the mRNAs of the CFTR, MRP and MDR proteins in the cells of the patients. According to a preferred embodiment, the products which can be used are therefore those whose administration leads to the appearance of, or to an increase in, the mRNAs which correspond to these proteins and therefore, in particular, to expression or overexpression of the MRP and/or MDR protein and/or, where appropriate, the CFTR protein itself.
It can also be particularly worthwhile to test, for example, products which are not sufficiently toxic toward cells to be active in anticancer therapy but which are nevertheless sufficiently active to give rise to a xe2x80x9cmultidrug resistancexe2x80x9d (MDR) phenomenon which can be taken advantage of within the context of the present invention.
All these compounds can be used under conditions which suffice to activate expression or overexpression of the MRP protein and/or the MDR protein and/or the CFTR protein.
Polychemotherapy by general, intermittent and sequential means is commonly used for the therapy of cancer. It is also possible to envisage using this type of treatment in accordance with the invention.
The invention therefore also relates to the use, for preparing a medicament which is intended for preventing and/or treating cystic fibrosis, of a product which contains at least one anticancer agent and/or macrolide as a combination product for simultaneous or separate use or for use which is staggered over time.
Preferably, said combination product will comprise an alkylating agent and an intercalating agent, and, even more preferably, the alkylating agent is ifosfamide and the intercalating agent is epirubicin.
The products which can be used in accordance with the invention are preferably administered together with a glutathione precursor, for simultaneous or separate use or for use which is staggered over time. In fact, it has often been observed that patients suffering from cystic fibrosis are deficient in glutathione and that administration of a glutathione precursor promotes an increase in the glutathione level. When the administered products are anticancer products which induce expression or overexpression of the MRP protein, the joint administration of a glutathione precursor is particularly desirable since the activity of the MRP protein depends on glutathione. Examples of glutathione precursors which may be mentioned are N-acetylcysteine (for example that marketed under the name Mucomyst) and N-acetyllysine.
The present invention also includes a second aspect. According to this aspect, a compound which directly replaces the defective CFTR protein, by playing the role of glutathione transporter, is administered to the patient.
The invention therefore also relates to a glutathione transporter compound as a medicament for secondarily preventing, and/or treating, cystic fibrosis. Some authors have already cloned a glutathione transporter compound into a rat liver (Yi et al., PNAS vol. 92, No. 5, 1995, 1495-1499). However, the possibility of a link with cystic fibrosis has never been considered.
Preferably this compound will be a fragment of a glutathione transporter ABC compound, in particular a fragment of the CFTR protein which comprises the NBF1 domain and, more specifically, a fragment which comprises the potential glutathione-binding site which is identified by reference to FIG. 3 in the examples which follow. The fragment is preferably a fragment which contains the residues I448-Q452, S478-K481, M498-I506, A566-L568, A596-T599, which are located in loops which are situated at the N-terminal ends of xcex2-pleated sheets 1 to 3 of the NBF1 domain. The joint administration of a glutathione precursor is also advantageous.
Finally, the invention relates to the application of the treatment according to the invention for treating rheumatoid arthritis or particular forms of asthma as well. These inflammatory pathologies are also likely to be initially caused by a defect in glutathione transport. Furthermore, they occur frequently in patients suffering from cystic fibrosis or in cystic fibrosis heterozygotes which only carry one modified allele out of the two for the CFTR gene.